Method of inhibiting cancer growth

ABSTRACT

The invention is a method of inhibiting a sarcoma, such as Kaposi&#39;s sarcoma, in a mammal. The method employs 6-demethyl-6-deoxy-4-de(dimethylamino)tetracycline (CMT-3.)

This is a continuation-in-part of U.S. application Ser. No. 09/007,645,filed on Jan. 15, 1998, now U.S. Pat. No. 6,100,248, which is acontinuation-in-part of U.S. application Ser. No. 08/783,655, filed onJan. 15, 1997, now U.S. Pat. No. 5,837,696 both of which areincorporated herein by reference.

This invention was made with Government support under Grant No.R37-DE03987 awarded by the National Institutes of Health through theNational Institute of Dental Research and Grant No. R29-CA61038 awardedby the National Institutes of Health. The Government has certain rightsin the invention.

BACKGROUND OF THE INVENTION

The invention relates to methods of reducing cancer growth in biologicalsystems. More specifically, the invention relates to the inhibition ofsolid tumor invasiveness and metastasis in mammals.

Cancer, in all of its myriad manifestations, remains a devastatingscourge upon mankind. While progress in preventing and treating cancerhas been made, including particular success against Hodgkin's lymphomaand certain other forms, many types of cancer remain substantiallyimpervious to prevailing treatment protocols. Typically, cancer istreated by chemotherapy, in which highly toxic chemicals are given tothe patient, or by radiotherapy, in which toxic doses of radiation aredirected at the patient. While commonly effective to kill huge numbersof cancer cells, these “cytotoxic” treatments also kill extraordinarynumbers of healthy cells, causing the patient to experience acutedebilitating symptoms including nausea, diarrhea, hypersensitivity tolight, hair loss, etc. The side effects of these cytotoxic compoundslimits the frequency and dosage at which they can be administered. Suchdisabling side effects can be mitigated to some degree by usingcompounds that selectively target cycling cells, i.e., interfering withDNA replication or other growth processes in cells that are activelyreproducing. Since cancer cells are characterized by their extraordinaryability to proliferate, such protocols preferentially kill a largerproportion of cancer cells in comparison to healthy cells, butcytotoxicity and ancillary sickness remains a problem.

Other more recent developments include efforts to develop monoclonalantibodies specific for oncogenes or HLA specificities, to identifycancer cells with great precision. However, these procedures are veryexpensive and extremely procedurally elaborate, yet still fail toproduce the desired efficacy. Indeed, such procedures have been reportedto be effective in only a small subpopulation of treated patients.

The area of cancer research concerned with the mechanisms of tumor cellinvasion has benefited greatly from the conceptual framework proposed byLiotta and colleagues (see, e.g., Yamamoto et al. (1996); Emmert-Buck etal. (1994). This model describes the invasive process as a logicalprogression of events involving three discernible stages: attachment oftumor cells to an extracellular matrix (ECM), proteolytic digestion ofthe matrix, and movement of cells through the proteolytically degradedbarrier. A key factor in this process is the regulation of the matrixmetalloproteinases (MMPs; including gelatinases A and B; MMP-2 andMMP-9, respectively, and MMP-3 (Lokeshwar et al. 1993a)), that play amajor role in the degradation of the ECM during invasion.

Tetracycline and a number of its chemical relatives form a particularlysuccessful class of antibiotics. Certain of the tetracycline compounds,including tetracycline itself, as well as sporocycline, etc., are broadspectrum antibiotics, having utility against a wide variety of bacteria.The parent compound, tetracycline, has the following general structure:

The numbering system for the multiple ring nucleus is as follows:

Tetracycline, as well as the 5-OH (terramycin) and 7-Cl (aureomycin)derivatives, exist in nature, and are all well known antibiotics.Semisynthetic derivatives such as 7-dimethylamino-tetracycline(minocycline) and 6α-deoxy-5-hydroxy-tetracycline (doxycycline) are alsoknown antibiotics. Natural tetracyclines may be modified without losingtheir antibiotic properties, although certain elements of the structuremust be retained to do so. The modifications that may and may not bemade to the basic tetracycline structure have been reviewed by Mitscher(1978). According to Mitscher, modification at positions 5-9 of thetetracycline ring system can be made without causing the complete lossof antibiotic properties.

However, changes to the basic structure of the ring system, orreplacement of substituents at positions 1-4 or 10-12, generally lead tosynthetic tetracyclines with substantially less, or essentially no,antibacterial activity. For example, 4-de(dimethylamino)tetracycline iscommonly considered to be a non-antibacterial tetracycline.

More recently, it has been established that tetracyclines, which arerapidly absorbed and have a prolonged plasma half-life, exert biologicaleffects independent of their antimicrobial activity (Golub et al. 1991,Golub et al. 1992, Uitto et al. 1994). Such effects include inhibitionof matrix metalloproteinases (abbreviated “MMPs”), includingcollagenases (MMP-1; MMP-8; MMP-13) and gelatinases (MMP-2; MMP-9), aswell as prevention of pathologic tissue destruction (Golub et al. 1991).Recent studies have suggested that, in some systems, certaintetracyclines and inhibitors of metalloproteinases can inhibit tumorprogression (DeClerck et al. 1994) or angiogenesis (WIPO publication WO92/12717; Maragoudakis et al. 1994). Zucker et al. (1985) showed thatminocycline can inhibit melanoma cell activity in vitro. Sometetracyclines may exhibit cytotstatic effects against some tumors (Kroonet al. 1984; van den Bogert et al. 1986).

However, the use of tetracycline antibiotics, while generally effectivefor treating infection, can lead to undesirable side effects. Forexample, the long term administration of antibiotic tetracyclines canreduce or eliminate healthy microbial flora, such as intestinal flora,and can lead to the production of antibiotic resistant organisms or theovergrowth of yeast and fungi. Accordingly, chemically-modifiedtetracyclines, in which the antimicrobial activity is attenuated ordeleted, can be preferred for use in applications in whichanti-collagenolytic activity is indicated.

In view of the above considerations, it is clear that there is a need tosupplement existing methods of inhibiting cancer cell invasiveness andmetastasis. Current approaches rely on highly cytotoxic compounds thatcause ancillary debilitating sickness in patients, or use methodologythat is expensive, procedurally difficult, and unpredictable.

Accordingly, it is one of the purposes of this invention to overcome theabove limitations in cancer treatment, by providing a compound andmethod for inhibiting the growth processes characteristic of cancercells, including inhibiting invasiveness and metastasis, as well asinducing regression of primary tumors. In particular, it is desirable toidentify new anticancer compounds and methods that inhibit cancer growthspecifically and with relatively high activity, i.e., being active atdoses that are substantially free of harmful side effects.

SUMMARY OF THE INVENTION

It has now been discovered that these and other objectives can beachieved by the present invention, which provides a method forinhibiting the growth or development of cancer in a mammal by providinga chemically modified tetracycline to the mammal in an amount that iseffective to achieve the specified result.

In one embodiment, the invention is a method of inhibiting cancer growthin a mammal, comprising administering to the mammal a cancer-inhibitoryamount of a tetracycline compound selected from the group consisting of:

4-de(dimethylamino)tetracycline (CMT-1),

6-demethyl-6-deoxy-4-de(dimethylamino)tetracycline (CMT-3),

4-de(dimethylamino)-7-chlorotetracycline (CMT-4),

tetracycline pyrazole (CMT-5),

6α-deoxy-5-hydroxy-4-de(dimethylamino)tetracycline (CMT-8),

4-de(dimethylamino)-12α-deoxyanhydrotetracycline (CMT-9), and

4-de(dimethylamino)minocycline (CMT-10).

A highly preferred tetracycline compound is

6-demethyl-6-deoxy-4-dedimethylaminotetracycline (CMT-3)

The method is useful for inhibiting growth of cancers such ascarcinomas, e.g., carcinomas of the lung, prostate, breast, ovary,testes, or colon, as well as melanomas.

The method can comprise inhibiting cellular proliferation of the cancer,inhibiting invasiveness of the cancer, and/or inhibiting metastasis ofthe cancer.

The tetracycline compound can be administered in an amount sufficient tospecifically inhibit expression of a matrix metalloproteinase by cellsof the cancer or its activity in the extracellular matrix.

In a preferred aspect, the method is useful to inhibit a matrixmetalloproteinase which is a gelatinase, such as gelatinase A orgelatinase B.

The method can further comprise treating the mammal with an adjunctantineoplastic modality. The adjunct antineoplastic modality cancomprise chemotherapy, surgery, and/or radiotherapy.

In another embodiment, the invention is a method of inhibiting cancergrowth in a mammal, comprising administering to the mammal acancer-inhibitory amount of a tetracycline compound selected from thegroup consisting of:

4-de(dimethylamino)tetracycline (CMT-1),

tetracyclinonitrile (CMT-2),

6-demethyl-6-deoxy-4-de(dimethylamino)tetracycline (CMT-3),

4-de(dimethylamino)-7-chlorotetracycline (CMT-4), and

4-hydroxy-4-de(dimethylamino)tetracycline (CMT-6),

4-de(dimethylamino)-12α-deoxytetracycline (CMT-7),

6-α-deoxy-5-hydroxy-4-de(dimethylamino)tetracycline (CMT-8),

4-de(dimethylamino)-12α-deoxyanhydrotetracycline (CMT-9), and

4-de(dimethylamino)minocycline (CMT-10).

In another embodiment, the invention is a method of inhibitingproliferation of cancer cells, comprising contacting the cancer cellswith a proliferation-inhibitory amount of a tetracycline compoundselected from the group consisting of:

6-demethyl-6-deoxy-4-de(dimethylamino)tetracycline (CMT-3),

6-α-deoxy-5-hydroxy-4-de(dimethylamino)tetracycline (CMT-8),

4-de(dimethylamino)tetracycline (CMT-1),

4-hydroxy-4-de(dimethylamino)tetracycline (CMT-6), and

4-de(dimethylamino)-12α-deoxytetracycline (CMT-7).

Preferably, the tetracycline compound is6-demethyl-6-deoxy-4-de(dimethylamino)tetracycline (CMT-3) or6-α-deoxy-5-hydroxy-4-de(dimethylamino)tetracycline (CMT-8).

In another embodiment, the invention is a method of inhibiting theinvasive potential of cancer cells, comprising contacting the cancercells with an invasion-inhibitory amount of a tetracycline compoundselected from the group consisting of:

6-demethyl-6-deoxy-4-de(dimethylamino)tetracycline (CMT-3),

4-de(dimethylamino)tetracycline (CMT-1),

4-de(dimethylamino)-12-deoxytetracycline (CMT-7),

4-hydroxy-4-de(dimethylamino)tetracycline (CMT-6),

4-de(dimethylamino)-7-chlorotetracycline (CMT-4),

6-α-deoxy-5-hydroxy-4-de(dimethylamino)tetracycline (CMT-8), andtetracyclinonitrile (CMT-2).

Preferably, the tetracycline compound is6-demethyl-6-deoxy-4-de(dimethylamino)tetracycline (CMT-3).

In still another embodiment, the invention is a method of inhibiting themetastatic potential of cancer cells, comprising contacting the cancercells with a metastasis-inhibitory amount of a tetracycline compoundselected from the group consisting of:

4-de(dimethylamino)tetracycline (CMT-1) and

6-demethyl-6-deoxy-4-de(dimethylamino)tetracycline (CMT-3).

In yet another embodiment, the invention is a method of treating acancer condition characterized by excessive gelatinolytic activity,comprising administering to a mammal an amount of a tetracyclinecompound effective to inhibit excessive gelatinolytic activity.

In this embodiment, the cancer may be characterized by excessiveactivity of gelatinase A, and the tetracycline compound is selected fromthe group consisting of:

4-hydroxy-4-de(dimethylamino)tetracycline (CMT-6),

4-de(dimethylamino)-12α-deoxytetracycline (CMT-7),

6-demethyl4-de(dimethylamino)tetracycline (CMT-3),

4-de(dimethylamino)tetracycline (CMT-1),

6-α-deoxy-5-hydroxy-4-de(dimethylamino)tetracycline (CMT-8),

4-de(dimethylamino)-7-chlorotetracycline (CMT-4), and

tetracyclinonitrile (CMT-2).

More preferably, the tetracycline compound is

4-hydroxy-4-de(dimethylamino)tetracycline (CMT-6),

4-de(dimethylamino)-12α-deoxytetracycline (CMT-7), or

6-demethyl-6-deoxy-4-de(dimethylamino)tetracycline (CMT-3).

Alternatively, in this embodiment, the cancer condition may becharacterized by excessive activity of gelatinase B, and thetetracycline compound is selected from the group consisting of:

6-demethyl-6-deoxy-4-de(dimethylamino)tetracycline (CMT-3),

4-de(dimethylamino)-7-chlorotetracycline (CMT-4),

4-de(dimethylamino)tetracycline (CMT-1), and

4-hydroxy-4-de(dimethylamino)tetracycline (CMT-6).

More preferably, in this case the tetracycline compound is

6-demethyl-6-deoxy-4-de(dimethylamino)tetracycline (CMT-3) or

4-de(dimethylamino)-7-chlorotetracycline (CMT-4).

In yet another embodiment, the invention is a method of inhibiting tumorincidence in a mammal, comprising

(a) detecting in a biological sample from the mammal a gene product ormetabolite associated with predisposition to a cancer prior to observingany specific cancerous lesion; and

(b) administering to the mammal a tumor incidence-inhibiting amount of atetracycline compound selected from the group consisting of:

4-de(dimethylamino)tetracycline (CMT-1),

tetracyclinonitrile (CMT-2),

6-demethyl-6-deoxy-4-de(dimethylamino)tetracycline (CMT-3),

4-de(dimethylamino)-7-chlorotetracycline (CMT-4), and

4-hydroxy-4-de(dimethylamino)tetracycline (CMT-6),

4-de(dimethylamino)-12α-deoxytetracycline (CMT-7),

6-α-deoxy-5-hydroxy-4-de(dimethylamino)tetracycline (CMT-8),

4-de(dimethylamino)-12α-deoxyanhydrotetracycline (CMT-9), and

4-de(dimethylamino)minocycline (CMT-10).

In still another embodiment, the invention is a method of inhibitinggelatinolytic activity associated with a cancerous tumor in a mammal,comprising administering to the mammal an amount of a tetracyclinecompound effective to inhibit gelatinolytic activity.

The gelatinolytic activity may derive from the cancerous tumor, or itmay derived from normal tissue, or both. If normal tissue is involved,the normal tissue may be epithelial tissue or stromal tissue.

In yet another embodiment, the invention is a method of inhibitingcancer growth in a mammal, comprising topically administering to themammal a cancer-inhibitory amount of a tetracycline compound selectedfrom the group consisting of:

tetracyclinonitrile (CMT-2) and

4-hydroxy-4-dedimethylaminotetracycline (CMT-6).

In another embodiment, the invention is a method of killing cancercells, comprising contacting cancer cells with a cytotoxic amount of atetracycline compound selected from the group consisting of:

4-de(dimethylamino)tetracycline (CMT-1),

tetracyclinonitrile (CMT-2),

6-demethyl-6-deoxy-4-de(dimethylamino)tetracycline (CMT-3),

4-de(dimethylamino)-7-chlorotetracycline (CMT-4),

4-hydroxy-4-de(dimethylamino)tetracycline (CMT-6),

4-de(dimethylamino)-12α-deoxytetracycline (CMT-7),

6-α-deoxy-5-hydroxy-4-de(dimethylamino)tetracycline (CMT-8),

4-de(dimethylamino)-12α-deoxyanhydrotetracycline (CMT-9), and

4-de(dimethylamino)minocycline (CMT-10).

In this embodiment, the cancer cells can be cells of a sarcoma,including Kaposi's sarcoma, or of a carcinoma, such as anadenocarcinoma. For example, the method can be used to kill cells of acarcinoma of the prostate, breast, ovary, testis, lung, colon, orbreast. Preferably, the cancer cells are cells of a carcinoma of theprostate, and the tetracycline compound is6-demethyl-6-deoxy-4-de(dimethylamino)tetracycline (CMT-3).

In yet another embodiment, the invention is a method of inhibiting thegrowth of a cancer in a mammal, comprising administering to a mammalhaving a cancer an amount of a tetracycline compound sufficient toinduce differential cytotoxicity in cells of the cancer, wherein thetetracycline compound is selected from the group consisting of:

4-de(dimethylamino)tetracycline (CMT-1),

tetracyclinonitrile (CMT-2),

6-demethyl-6-deoxy-4-de(dimethylamino)tetracycline (CMT-3),

4-de(dimethylamino)-7-chlorotetracycline (CMT-4),

4-hydroxy-4-de(dimethylamino)tetracycline (CMT-6),

4-de(dimethylamino)-12α-deoxytetracycline (CMT-7),

6-α-deoxy-5-hydroxy-4-de(dimethylamino)tetracycline (CMT-8),

4-de(dimethylamino)-12α-deoxyanhydrotetracycline (CMT-9), and

4-de(dimethylamino)minocycline (CMT-10).

By means of the invention, a method of killing cancer cells orinhibiting cancer growth or metastasis is provided that further avoidsor mitigates side effects commonly associated with antineoplasticchemotherapeutic regimens. These and other advantages of the presentinvention will be appreciated from the detailed description and examplesset forth hereinbelow. The detailed description and examples enhance theunderstanding of the invention, but are not intended to limit the scopeof the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

Preferred embodiments of the invention have been chosen for purposes ofillustration and description, but are not intended in any way torestrict the scope of the invention. The preferred embodiments ofcertain aspects of the invention are shown in the accompanying drawings,wherein:

FIGS. 1A to 1D are four electrophoretograms depicting zymographicanalysis of conditioned media obtained from prostate cancer cells invitro illustrating the effects of CMT-3 and doxycycline on MMP activity:TSU PR1 prostate tumor cells treated with CMT-3 (FIG. 1A) or doxycycline(FIG. 1B); MAT LyLu prostate tumor cells treated with CMT-3 (FIG. 1C) ordoxycycline (FIG. 1D).

FIGS. 2A and 2C are graphs illustrating the in vitro dosage-dependentinhibition of cellular proliferation by tetracycline derivatives in:LNCaP tumor cells (FIG. 2A); TSU PR1 tumor cells (FIG. 2B); and MAT LyLutumor cells (FIG. 2C).

FIGS. 3A to 3D are graphs illustrating the in vitro dosage-dependentinhibition of cellular proliferation by tetracycline derivatives in:DU-145 tumor cells (FIG. 3A); PC-3 tumor cells (FIG. 3B); BPH-1non-tumorigenic prostate epithelial cells (FIG. 3C); and FHS733 normalhuman fibroblasts (FIG. 3D).

FIGS. 4A and 4B are graphs illustrating the in vitro dosage-dependentinduction of cytotoxicity by tetracycline derivatives in Dunning MATLyLu tumor cells (a rat prostate cancer model) at: 24 hr exposure (FIG.4A); and 48 hr exposure (FIG. 4B).

FIG. 5A is a histogram providing a comparative illustration of thecapacity of CMT compounds to inhibit invasiveness of TSU PRI tumor cells(a human prostate cancer cell line) in vitro; FIG. 5B is a histogramproviding a comparative illustration of the capacity of CMT compounds toinhibit invasiveness of MAT LyLu tumor cells in vitro.

FIG. 6 is a histogram illustrating the inhibition of MAT LyLu tumormetastasis by tetracycline compounds.

FIG. 7A is a histogram demonstrating the capacity of CMT-3 to reducetumor incidence following implantation of MAT LyLu tumor cells into testanimals;

FIG. 7B is a histogram illustrating the inhibition of MAT LyLu tumormetastasis by tetracycline compounds.

FIG. 8 is a histogram illustrating the relationship between inhibitionof melanoma cell invasivity and dosage of chemically modifiedtetracyclines.

FIGS. 9A to 9D are graphs illustrating dosage-dependent cytotoxicity ofCMT-3 in: normal prostate stromal cells (FIG. 9A); LNCaP tumor cells(FIG. 9B); PC-3 tumor cells (FIG. 9C); and DU-145 tumor cells (FIG. 9D).

FIG. 10A is a histogram showing dosage-dependent tetracycline-inducedgeneration of nucleosomes by MAT LyLu cells; FIG. 10B showstime-dependent CMT-3-induced generation of nucleosomes by MAT LyLucells.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

In one embodiment, the present invention is directed to a method forinhibiting cancer growth, including processes of cellular proliferation,invasiveness, and metastasis in biological systems. The method includesthe use of a tetracycline compound as an inhibitor of cancer growth.Preferably, the method is employed to inhibit or reduce cancer cellproliferation, invasiveness, metastasis, or tumor incidence in livinganimals, such as mammals. The method is also readily adaptable for usein assay systems, e.g., assaying cancer cell growth and propertiesthereof.

The compounds useful according to the invention possess a desirable butunusual combination of physicochemical properties, including activity,bioavailability, solubility, and reduction of side effects. Theseproperties render the compounds particularly desirable for the treatmentof cancer.

Such compounds include, for example, those that lack the dimethylaminogroup at position 4 of the tetracycline ring structure, e.g.,

4-de(dimethylamino)tetracycline (CMT-1),

6-demethyl-6-deoxy-4-de(dimethylamino)tetracycline (CMT-3),

7-chloro-4-de(dimethylamino)tetracycline (CMT-4),

4-hydroxy-4-de(dimethylamino)tetracycline (CMT-6),

4-de(dimethylamino)-12α-deoxytetracycline (CMT-7),

6-deoxy-5α-hydroxy-4-de(dimethylamino)tetracycline (CMT-8),

4-dedimethylamino-12α-deoxyanhydrotetracycline (CMT-9),

7-dimethylamino-6-demethyl-6-deoxy-4-de(dimethylamino)tetracycline(CMT-10),

4-de(dimethylamino)-5-oxytetracycline,

5α,6-anhydro-4-hydroxy-4-de(dimethylamino)tetracycline,

4-de(dimethylamino)-11-hydroxy-12α-deoxytetracycline,12α-deoxy-4-deoxy-4-de(dimethylamino)tetracycline, and12α,4α-anhydro-4-de(dimethylamino)tetracycline.

Further examples of tetracyclines modified for reduced antimicrobialactivity include 6-α-benzylthiomethylenetetracycline, themono-N-alkylated amide of tetracycline, 6-fluoro-6-demethyltetracycline,11α-chlorotetracycline, tetracyclinonitrile (CMT-2), and tetracyclinepyrazole (CMT-5).

The preferred tetracycline compounds include:

4-de(dimethylamino)tetracycline (CMT-1),

6-demethyl-6-deoxy-4-de(dimethylamino)tetracycline (CMT-3),

4-de(dimethylamino)-7-chlorotetracycline (CMT-4), and

6-α-deoxy-5-hydroxy-4-de(dimethylamino)tetracycline (CMT-8).

The most preferred tetracycline compound is6-demethyl-6-deoxy-4-de(dimethylamino)tetracycline (CMT-3). Combinationsof these compounds can be employed. Doxycycline is not included withinthe invention.

These compounds exhibit their cancer-inhibitory properties atconcentrations that lead to fewer side effects than those of knownchemotherapeutic agents, and in some cases are substantially free ofside effects. For example, the useful concentrations of the compounds donot present any significant antimicrobial activity. These tetracyclinecompounds are useful for extended treatment protocols, where othercompounds would exhibit undesirable side-effects. In addition, it isbelieved that the properties of hydrophilicity and hydrophobicity arewell balanced in these compounds, enhancing their utility both in vitroand especially in vivo, while other compounds lacking such balance areof substantially less utility. Specifically, the compounds have anappropriate degree of solubility in aqueous media to permit absorptionand bioavailability in the body, while also having a degree ofsolubility in lipids to permit traversal of the cell membrane to aputative site of action. The compounds are maximally effective if theycan be delivered to the site of the tumor and are able to enter thetumor cells.

In the treatment of certain localized cancers, the degree ofhydrophilicity of the non-antimicrobial tetracycline compound can be oflesser importance. Such compounds as tetracyclinonitrile (CMT-2) and4-hydroxy-4-de(dimethylamino)tetracycline (CMT-6), which have lowsolubility in aqueous systems, can be used in direct or topicaltreatment of skin cancers, e.g., melanoma or basal cell carcinoma, or byimplantation into the brain to topically treat brain cancer. Animalexperiments, in which adult rats are orally gavaged with these two CMTs,have shown no detectable blood levels of these compounds, indicating alack of systemic absorption and/or extraordinarily rapid excretion.

This embodiment of the invention has been developed based on theunexpected observation by Applicants that certain tetracyclinecompounds, chemically modified to eliminate substantially allantimicrobial activity, are effective to inhibit the proliferation,invasiveness, or metastasis of cancer cells in vitro, as well as invivo. Of these, one especially preferred CMT, i.e.,6-demethyl-6-deoxy-4-de(dimethylamino)tetracycline (also referred to as“CMT-3”), appears to possess an excellent balance of properties, in thatit is shown to possess unusually strong activity in inhibiting thecancer growth, including proliferation, invasiveness, or metastasis ofcancer cells. Another advantage of CMT-3 is that it has an unexpectedlylong serum half-life (approximately 28 hr). Therefore, CMT-3 may onlyrequire periodic administration, e.g., once or twice per week.

In another embodiment, the method of the invention is effective toinhibit the enzymatic activity of matrix metalloproteinases, such ascollagenases and gelatinases, associated with cancerous tumors inmammals. The gelatinolytic activity capable of inhibition may derivefrom gelatinase expression by the cancerous tumor or from normal, i.e.,non-cancerous, tissue. In particular, the gelatinase activity may bederived from such normal tissues as epithelial tissue or stromal tissue.More preferably, the method can be used to inhibit excessivegelatinolytic activity associated with such tumors. The gelatinasescapable of inhibition include gelatinase A (also known as 72 kDa typegelatinase; MMP-2; type IV collagenase); and gelatinase B (also known as92 kDa type gelatinase; MMP-9; type V collagenase). This inhibition ofobserved gelatinolytic activity may be due to inhibition of MMPactivity, down-regulation of MMP expression, or some other interferencewith the physiology associated with these gelatinases, such asinhibition of activation of the precursor form of the enzyme,pro-gelatinase (or pro-MMP).

While Applicants do not wish to be bound by any particular mechanismwith respect to the present invention, Applicants were surprised to findthat CMTs can act, inter alia, by a fundamentally unknown mechanism inthe context of cancer. Applicants have discovered that the chemicallymodified tetracyclines decrease the level of expression of(“down-regulate”) metalloproteinases normally associated with cancer.For example, it has been found that CMT-3 reduces expression ofgelatinase A by human colorectal cancer cells and inhibits expression ofgelatinase B by human breast cancer cells. Applicants believe that thisobservation carries significant therapeutic implications for cancertreatment. Applicants also understand that these CMTs and otherchemically and functionally related compounds would be useful forinhibiting the consequences of other diseases characterized by excessivegelatinase expression or activity.

The invention includes a method of inducing cytotoxicity (cell killing)in cancer cells or reducing the viability of cancer cells. Thecytotoxicity of tetracycline compounds can be exploited preferablyagainst sarcomas, e.g. Kaposi's sarcoma, or carcinomas, e.g.,adenocarcinomas. For example, the invention can be used to inducecytotoxicity in cells of carcinomas of the prostate, breast, ovary,testis, lung, colon, or breast. The mechanism by which this cytotoxicityoccurs is not completely understood, but the selective killing of thecancer cells can occur through apoptosis, necrosis, another mechanism,or a combination of mechanisms.

The killing of cancer cells can occur with less cytotoxicity to normalcells or tissues than is found with conventional cytotoxic therapeutics,preferably without substantial cytotoxicity to normal cells or tissues.For example, it has been unexpectedly observed that a tetracycline,e.g., CMT-3, can induce cytotoxicity in cancer cells while producinglittle or substantially no cytotoxicity in normal cells. Thus, unlikeconventional cytotoxic anticancer therapeutics, which typically kill allgrowing cells, CMT-3 can produce differential cytotoxicity: tumor cellsare selectively killed whereas normal cells are spared. Thus, in anotherembodiment, the invention is a method for inducing differentialcytotoxicity in cancer cells relative to normal cells or tissue. Thisunexpected differential cytotoxicity associated with the tetracyclinecompounds occurs as a result of apoptosis, necrosis, another mechanism,or a combination of such mechanisms.

The data presented in the examples below, reveal that cancer cellstreated with these compounds results in a decrease in extracellulargelatinolytic activity, a corresponding dose-dependent decrease in thecells' in vitro invasive potential, and a decrease in the cells'metastatic ability in vivo. Moreover, the compounds can induce killingof tumor cells, and can do so while being substantially non-cytotoxic tonormal tissues. Accordingly, these chemically-modified tetracyclines canbe used to suppress the formation and magnitude of metastases associatedwith certain cancers, used as an adjunct to other treatment regimens,and lead to greater efficacy in the treatment of metastatic cancers.

The cancers treatable by means of the present invention occur inmammals. Mammals include, for example, humans, as well as pet animalssuch as dogs and cats, laboratory animals such as rats and mice, andfarm animals such as horses and cows.

Tumors or neoplasms include new growths of tissue in which themultiplication of cells is uncontrolled and progressive. Some suchgrowths are benign, but others are termed “malignant,” leading to deathof the organism. Malignant neoplasms or “cancers” are distinguished frombenign growths in that, in addition to exhibiting aggressive cellularproliferation, they invade surrounding tissues and metastasize.Moreover, malignant neoplasms are characterized in that they show agreater loss of differentiation (greater “dedifferentiation”), and oftheir organization relative to one another and their surroundingtissues. This property is also called “anaplasia.”

Neoplasms treatable by the present invention include all solid tumors,i.e., carcinomas and sarcomas, including Kaposi's sarcoma. Carcinomasinclude those malignant neoplasms derived from epithelial cells whichtend to infiltrate (invade) the surrounding tissues and give rise tometastases. Adenocarcinomas are carcinomas derived from glandular tissueor in which the tumor cells form recognizable glandular structures.Sarcoma, including Kaposi's sarcoma broadly include tumors whose cellsare embedded in a fibrillar or homogeneous substance like embryonicconnective tissue.

The invention is particularly illustrated herein in reference totreatment of certain types of experimentally defined cancers. In theseillustrative treatments, standard state-of-the-art in vitro and in vivomodels have been used. These methods can be used to identify agents thatcan be expected to be efficacious in in vivo treatment regimens.However, it will be understood that the method of the invention is notlimited to the treatment of these tumor types, but extends to any solidtumor derived from any organ system. Cancers whose invasiveness ormetastasis is associated with MMP expression, particularly gelatinaseexpression, are especially susceptible to being inhibited or eveninduced to regress by means of the invention.

Thus, the treatable cancers include, for example, colon cancer, bladdercancer, breast cancer, melanoma, ovarian carcinoma, prostatic carcinoma,or lung cancer, and a variety of other cancers as well. The invention isespecially useful in the inhibition of cancer growth in adenocarcinomas,including, for example, those of the prostate, breast, kidney, ovary,testes, and colon. The invention is further useful against melanomas,which derive from the melanocytic system in the skin and other organs.

The method involves providing or administering a tetracycline compoundin an amount that is effective for reducing cancer cell growth, i.e.,cellular proliferation, invasiveness, metastasis, or tumor incidence ina mammal. The inhibition may result from inhibition of MMP activity,down-regulation of MMP expression, some other mechanism, or acombination of mechanisms. For example, Applicants have found that CMT-3inhibits the expression of MMP-2 and MMP-9 in cancer cells in vitro. Itis believed that all solid cancer types that express MMPs or thatexhibit invasive or metastatic properties can be treated by the methodof the invention. In some cases, the incidence or development of tumorfoci can be inhibited or substantially prevented from occurring.Therefore, the method can be used as a prophylactic treatment, e.g., byadministering the tetracycline compound to a mammal after detection of agene product or metabolite associated with predisposition to a cancerbut before any specific cancerous lesion is detected. Alternatively, thetetracycline compounds are useful for preventing cancer recurrence, forexample, to treat residual cancer following surgical resection orradiation therapy. The tetracycline compounds useful according to theinvention are especially advantageous as they are substantiallynon-toxic compared to other cancer treatments.

The effect occurs over a wide range of concentrations, including atconcentrations that are extraordinarily low. The amount of thetetracycline compound used according to the invention is an amount thatis effectively inhibitory of cancer growth. An amount of a tetracyclinecompound is effectively inhibitory to cancer growth if it significantlyreduces cellular proliferation or the potential of invasiveness ormetastasis. Proliferation refers to the capacity of a tumor to increaseits volume through cell division, typically measured as the “doublingrate.” The inhibition of cellular proliferation by the present methodmeans that the rate of growth is decreased. In some cases, the methodcan actually induce regression or diminution of tumor mass, if the rateof replenishment of the tumor cells through cell division is exceeded bythe rate of cell death. Invasiveness refers to the potential of a tumoror tumor cells to invade other tissues, typically by breaking down theextracellular matrix of those tissues. Metastasis refers to thepotential of a tumor or tumor cells to establish new tumor foci at sitesdistant from the primary site where the tumor began. Typically,metastasis proceeds by individual cells or groups of cells breaking offfrom the primary tumor and migrating, e.g., through the blood or lymph,to establish a new tumor focus in another tissue or organ. One locuscommon in tumor metastasis is in the lung, where the very finevasculature of the lung tissue can often catch circulating tumor cells,permitting the establishment of a tumor focus therein. Some types oftumors metastasize to specific types of tissues. For example, prostateadenocarcinomas can metastasize to bone with great specificity. The datapresented herein provide evidence that the method of the invention iscapable of inhibiting cancer growth and recurrence as defined by any orall of these parameters.

Preferably, the tetracycline compound is provided in an amount that haslittle or no antimicrobial activity. A tetracycline compound is noteffectively antimicrobial if it does not significantly prevent thegrowth of microbes. Accordingly, the method can beneficially employ atetracycline compound that has been modified chemically to reduce oreliminate its antimicrobial properties. The use of suchchemically-modified tetracyclines is preferred in the present inventionsince they can be used at higher levels than antimicrobialtetracyclines, while avoiding certain disadvantages, such as theindiscriminate killing of beneficial microbes, and the emergence ofresistant microbes, which often accompanies the use of antimicrobial orantibacterial amounts of such compounds over prolonged periods of time.

The tetracycline compounds useful according to the method of theinvention appear to exhibit their beneficial effect in a dose-dependentmanner. Thus, within broad limits, administration of larger quantitiesof a tetracycline compound has been observed to inhibit cancer cellgrowth or invasiveness to a greater degree than does administration of asmaller amount. Moreover, efficacy has been observed at dosages belowthe level at which toxicity is seen in normal cells or at the organismallevel. Accordingly, one of the advantages of the invention is that thedebilitating side effects usually attendant upon conventional cytotoxiccancer treatments are reduced, and preferably avoided.

The maximal dosage for a subject is the highest dosage that does notcause undesirable or intolerable side effects. For example, thetetracycline compound(s) can be administered in an amount of from about0.1 mg/kg/day to about 30 mg/kg/day, and preferably from about 1mg/kg/day to about 18 mg/kg/day. For the purpose of the presentinvention, side effects may include clinically significant antimicrobialor antibacterial activity, as well as toxic effects. For example, a dosein excess of about 50 mg/kg/day would likely produce side effects inmost mammals, including humans. In any event, the practitioner is guidedby skill and knowledge in the field, and the present invention includes,without limitation, dosages that are effective to achieve the describedphenomena.

The invention can also be practiced by including with the tetracyclinecompound one or more other anti-cancer chemotherapeutic agents, such asany conventional chemotherapeutic agent. The combination of thetetracycline compound with such other agents can potentiate thechemotherapeutic protocol. Numerous chemotherapeutic protocols willpresent themselves in the mind of the skilled practitioner as beingcapable of incorporation into the method of the invention. Anychemotherapeutic agent can be used, including alkylating agents,antimetabolites, hormones and antagonists, radioisotopes, as well asnatural products. For example, the non-anti-microbial tetracyclinecompound can be administered with antibiotics such as doxorubicin andother anthracycline analogs, nitrogen mustards such as cyclophosphamide,pyrimidine analogs such as 5-fluorouracil, cisplatin, hydroxyurea, taxoland its natural and synthetic derivatives, and the like. As anotherexample, in the case of mixed tumors, such as adenocarcinomas of thebreast and prostate, in which the tumors can includegonadotropin-dependent and gonadotropin-independent cells, thetetracycline can be administered in conjunction with leuprolide orgoserelin (synthetic peptide analogs of LH-RH). Other antineoplasticprotocols include the use of a tetracycline compound with anothertreatment modality, e.g., surgery, radiation, other chemotherapeuticagent, etc., referred to herein as “adjunct antineoplastic modalities.”Thus, the method of the invention can be employed with such conventionalregimens with the benefit of reducing side effects and enhancingefficacy.

The preferred pharmaceutical composition for use in the method of theinvention includes a combination of the tetracycline compound in asuitable pharmaceutical carrier (vehicle) or excipient as understood bypractitioners in the art.

Enteral administration is a preferred route of delivery of thetetracycline, and compositions including the tetracycline compound withappropriate diluents, carriers, and the like are readily formulated.Liquid or solid (e.g., tablets, gelatin capsules) formulations can beemployed. It is among the advantages of the invention that, in manysituations, the tetracycline compound can be delivered orally, asopposed to parenteral delivery (e.g., injection, infusion) which istypically required with conventional chemotherapeutic agents.

Parenteral use (e.g., intravenous, intramuscular, subcutaneousinjection) is also contemplated, and formulations using conventionaldiluents, carriers, etc., such as are known in the art can be employedto deliver the compound.

Alternatively, delivery of the tetracycline compound can include topicalapplication. Compositions deemed to be suited for such topical useinclude as gels, salves, lotions, ointments and the like. In the case oftumors having foci inside the body, e.g., brain tumors, the tetracyclinecompound can be delivered via a slow-release delivery vehicle, e.g., apolymeric material, surgically implanted at or near the lesion situs.

In developing the present invention, several chemically modifiedtetracyclines (CMTs) were tested for their effect (in comparison to acommercially available antibacterial tetracycline, doxycycline) ininhibiting cancer growth. This testing investigated the effect of thesecompounds on prostate cancer cell proliferation and invasive potentialin vitro, and on tumor growth and lung colonization of an in vivo tumormodel, the rat Dunning MAT LyLu. During in vitro experiments,antimicrobial Doxycycline and certain non-antimicrobial CMTs inhibitedthe cell proliferation of human prostate tumor cell lines (PC-3, DU-145,TSU PR1, and LNCaP), and the Dunning prostate tumor cells (IC₅₀=3-120μg/mL). Doxycycline and CMTs also inhibited invasive potential by 10% to90%, depending on the compound. CMT-3(6-deoxy-6-demethyl-4-de(dimethylamino)tetracycline) was the most potentof the tetracycline analogues, inhibiting invasive potential by 90% at 5μg/mL, a level of this drug readily achieved in vivo by oraladministration. Growth of the Dunning tumor at the primary site (s.c.)was not altered significantly in rats treated with either doxycycline orCMT-3 by oral gavage daily for 21 days following tumor implant. Therewas a significant decrease, however, in the number of lung metastases:28.9±15.4 sites/animal in the CMT-3-treated group versus 59.5±13.9sites/animal in controls (p<0.01), which is double the effect seen withdoxycycline at the same oral dose. Predosing the rats 7 days prior totumor implant resulted in a significant delay in tumor growth (46±9.3%,p<0.05) and a reduction in metastasis. In addition, tumor remission(inhibition of tumor incidence) occurred in the groups treated withCMT-3 (40 mg/kg). Treatment with doxycycline, however, did not result intumor remission. In addition, a 58±8% decrease in the number of lungmetastases was observed in the CMT-3-treated group versus a decrease of33±7.0% in the doxycycline group. No significant drug-induced morbiditywas observed in any of the experiments described herein. These resultsfurther substantiate the usefulness of CMT-3 for chemotherapeutictreatment to control tumor aggression and prevent metastasis.

In other experiments described hereinbelow, we examined the effect(s) ofdoxycycline and CMTs on extracellular levels of gelatinase A and Bactivity from a highly invasive and metastatic human melanoma cell lineC8161, and correlated these observations with changes in the cells'biological behavior in an in vitro invasion assay and in an in vivo SCIDmouse model. The results indicate that coincident with the ability ofthese compounds to differentially suppress extracellular levels ofgelatinase activity, C8161 cells treated with doxycycline and CMT-1,CMT-3, and CMT-6 were less invasive in vitro, in a dose dependent manner(3-50 μg/mL). Furthermore, data derived from the in vivo model indicatethat SCID mice dosed orally with CMT-1 and CMT-3 contained a reducednumber of lung metastases following intravenous injection of C8161 cellsvia tail vein inoculation.

The following examples are provided to assist in a further understandingof the invention. The particular materials and conditions employed areintended to be further illustrative of the invention and are notlimiting upon the reasonable scope thereof.

EXAMPLE 1A

Inhibition of Enzyme Expression in Cancer Cells

Two human cancer-derived cell lines were obtained from the American TypeCulture Collection (ATCC) in Rockville, Md. The cell lines included COLO205, a human colon cancer-derived cell line that expresses MMP-2 orgelatinase A, and E-10, a human breast cancer-derived cell line thatexpresses MMP-9 or gelatinase B.

Cells of each cell line were cultured in 75 cm² T-flasks in RPMI-1640(Gibco) with 10% heat-inactivated fetal bovine serum (FBS) containing100 units/mL penicillin and 100 μg/mL streptomycin. The cells were fedevery two days, and passaged every week. The cells were grown to 80-90%confluence, and then the FBS-containing medium was replaced with aserum-free medium (SFM). CMT-3 (CollaGenex Pharmaceuticals, Inc.,Newtown, Pa.) was added in several concentrations to the cells in SFM.Conditioned medium was collected after 24 hr, centrifuged to remove celldebris, and then assayed for MMP protein expression by Western blotusing a conventional technique, and scanning the blots with a laserdensitometer. The data obtained are summarized in Table 1, below.

TABLE 1 Percent Inhibition of MMP Protein Expression in Cancer CellLines by CMT-3 CMT-3 Concentration Cancer Cell Line 0 μM 10 μM 20 μMCOLO 205 (expressing MMP-2) — 13.3% 66.9% E-10 (expressing MMP-9) —45.3% 60.8%

These data clearly show dose-dependent activity of CMT-3 in inhibitingexpression of two different MMPs in two different types of cancer cells.It is believed that the inhibition of MMP expression inhibits theability of these cancer cells to degrade the extracellular matrix ofhealthy tissues thereby limiting the cancer's ability to invade suchhealthy tissues. The inhibition of cancer cell proliferation in vitroand cancer metastasis in vivo is demonstrated in several followingexamples.

EXAMPLE 1B

Inhibition of Matrix Metalloproteinase Activity in Cancer Cells

The effects of CMT-3 and doxycycline on gelatinase activity orexpression by prostate cancer cells were tested. First, we tested thecapacity of CMT-3 and doxycycline to inhibit MMP secretion into culturemedium. Gelatinase activity was measured using a method adapted from themethod of Dean and Woessner (1985). Conditioned culture medium wascollected from MAT LyLu and TSU PR1 cells treated with CMT-3 ordoxycycline for two days. The serum-free culture medium comprised RPMI1640 basal medium containing insulin (5 μg/mL), transferrin (5 μg/mL),selenious acid (5 μg/mL), and gentamicin (2 μg/mL). Initial assays ofthese conditioned media showed the presence of TIMPs which interferedwith the MMP assay. Therefore the conditioned media were chemicallyreduced (1 mM dithiothreitol) and alkylated (1 mM iodoacetamide) eachfor 30 min at 37° C., and then dialyzed to destroy endogenous TIMPs.(Dean et al. 1987; Woessner 1991.) The dialyzed medium was assayed forgelatinase activity following activation of latent MMPs with 1 mMp-aminophenyl mercuric acetate (APMA) for 30 min at 37° C. The assay mixwas 25 μg ³H-gelatin (prepared from heat-denatured ³H-acetylatedacid-soluble collagen), 0.1 mL of processed culture-conditioned medium,1 mM phenylmethyl sulfonyl fluoride (PMSF) in a total volume of 0.5 mLof an assay buffer (20 mM Tris-HCl, pH 7.4, 30 mM NaCl, 0.005% Brij35,10 mM CaCl₂, 2 μM ZnSO₄, and 0.02% NaN₃). In some cases, 1 mM CaCl₂(instead of 10 mM) was used. Blanks were obtained by adding 1 mM1,10-phenanthroline. Doxycycline or CMT-3 were added to the assaymixture following activation of the latent MMPs and just before addingthe substrate ³H-gelatin.

In addition, the ability of doxycycline or CMT-3 to inhibit APMAactivation of MMPs was tested. Processed culture-conditioned medium wasincubated with the drugs for 1 hr before adding 1 mM APMA, and theincubation was continued thereafter for 2 hr. ³H-gelatin was then addedand the assy was continued for 4 hr. All assays were performed induplicate tubes, and repeated at least twice.

MMP activity in the culture media was examined at 1 mM and 10 mM CaCl₂,with no change in [Zn²⁺] in the assay buffer. We employed 1 mM CaCl₂, asthis concentration is closer to physiological concentration, and 10 mMCaCl₂, as it is reported to be the optimum concentration in in vitroassays (Woessner 1991). As is shown in Table 2, both CMT-3 anddoxycycline inhibited in vitro activated gelatinases. At 1 mM CaCl₂, the50% inhibition dose (IC₅₀) of CMT-3 was 0.5 μM, while at 10 mM CaCl₂,the IC₅₀ for CMT-3 was ˜1.5 μM. By contrast, at 10 mM CaCl₂, the IC₅₀for doxycycline was 5.0 μM. Furthermore, both of these drugs stronglyinhibited activation of MMPs by p-aminophenyl mercuric acetate; the IC₅₀for CMT-3 was 1.0 μg/mL (2.2 μM), and for doxycycline was 2.5 μg/mL (5μM) at 10 mM CaCl₂.

TABLE 2 Enzyme Inhibition by CMT-3 and Doxycycline in Prostate CancerCells Drug Con- Percent Inhibition centration CMT-3 Doxycycline (μM) 1mM CaCl₂ 10 mM CaCl₂ 1 mM CaCl₂ 10 mM CaCl₂ 0.25 ND 31.5 ± 1.2 ND ND0.50 84.8 ± 10.1 37.8 ± 7.5^(b) 54.8 ± 0.6^(a) ND 1.0 96.7 ± 4.6^(a)45.1 ± 2.9  77.4 0.0 2.0 ND 51.2 ± 2.6  ND ND 5.0 97.2 ± 2.9  52.4 ±3.5  ND 64.3 ± 19.5 10 100 69.9 ± 10.5 91.2 ± 3.3  75.5 ± 3.3  20 ND78.9 ± 11.4 ND 85.1 ± 3.5  50 ND 94.2 ± 3.3  100 93.2 ± 2.7  100 ND NDND 91.2 ± 2.6  ^(a)Total gelatinase activity in the presence of 1 mMCaCl₂, without inhibitors (control) was 18.95 ± 3.18 ng [³H]gelatindigested/min/mL of the dialyzed medium. ^(b)Total gelatinase activity inthe presence of 10 mM CaCl₂, without inhibitors (control) was 48.92 ±2.7 ng/min/mL.

Next we examined whether CMT-3 and doxycycline differentially affect theproduction of two major classes of gelatinases: gelatinase A (MMP-2) andgelatinase B (MMP-9). Confluent cultures of TSU PR1 and MAT LyLu cellswere incubated with various concentrations of doxycycline and CMT-3 for48 hours in serum-free medium. The conditioned media were then analyzedfor MMPs by zymography as described below.

Conditioned media were collected from the cultures that had been treatedwith doxycycline or CMT-3 for 2 days. Media were then incubated withSDS-gel electrophoresis sample buffer for 30 min at room temperature,and then analyzed by gel electrophoresis on SDS-polyacrylamide gel (8%)containing gelatin (1 mg/mL). Following electrophoresis, the gels werewashed twice with 0.25% TRITON® X100 for 30 min each, and incubated for18 hr at 37° C. in an MMP digestion buffer comprising 20 mM Tris-HCl, pH7.4, containing 30 mM NaCl, 1 mM PMSF, 10 mM CaCl₂, 2 μM ZnSO₄, 0.005%Brij35, and 0.02 NaN₃ (Lokeshwar 1993a). After incubation, the gels werebriefly rinsed in distilled water and stained with 0.25% Coomassiebrilliant blue R250 prepared in 40% isopropanol solution for 1 hr. Gelswere destained with 7% acetic acid. the locations of MMPs in the gelswere visible as clear areas on a blue background, indicative of digestedgelatin.

The results are shown in FIGS. 1A to 1D. The conditioned media derivedfrom both cell lines contained predominantly MMP-2 and MMP-9. The TSUPR1 medium contained predominantly the latent forms of these two enzymes(FIGS. 1A and 1B), whereas the MAT LyLu medium contained activated MMP-2but little MMP-9 (FIGS. 1C and 1D).

Both drugs produced a dose-dependent decrease in MMP-2 levels. CMT-3,however, decreased MMP-2 and MMP-9 levels at a lower concentration thandid doxycycline. Moreover, MMP-9 levels did not decrease significantlywith an increase in doxycycline concentration. The decrease in MMPlevels with increasing concentration of the two tetracycline compoundswas specific to MMPs, because all lanes were loaded with the same totalamount of protein.

EXAMPLES 2-6

In Examples 2-6, below, the following materials and methods wereemployed:

Reagents: Chemically modified tetracyclines were prepared according toknown methods. The synthesis of various CMTs is extensively documented.See, e.g., Mitscher (1978). The following CMTs were investigated:4-de(dimethylamino)tetracycline (CMT-1), tetracyclinonitrile (CMT-2),6-demethyl-6-deoxy-4-de(dimethylamino)tetracycline (CMT-3),4-de(dimethylamino)-7-chlorotetracycline (CMT-4),4-hydroxy-4-de(dimethylamino)tetracycline (CMT-6),4-de(dimethylamino)-12α-deoxytetracycline (CMT-7), and6-α-deoxy-5-hydroxy-4-de(dimethylamino)tetracycline (CMT-8). Highlypurified CMT-3 (93-98%) used for animal studies was supplied byCollaGenex, Inc., Newtown, Pa. Doxycycline was purchased from SigmaChemical Co., St. Louis, Mo. Matrigel, a solubilized preparation oftumor basement membrane, was obtained from Collaborative Research,Bedford, Mass. Boyden Chemotaxis assay chambers (Transwells) wereobtained from Costar/Corning Corp., Boston, Mass. All other reagentswere from Sigma Chemical Co.

Cells and Tumor Lines: Human prostate cancer cell lines: PC-3, DU-145,and LNCaP were obtained from American Type Culture Collection,Rockville, Md. TSU PR1 and ALVA 101, human metastatic prostatic cancercell lines, and BPH-1, a non-tumorigenic prostate cell line were alsoused (Lokeshwar 1995b, 1996; Hayward (1995). Cultures were maintained incomplete medium composed of RPMI-1640 medium with 10% fetal bovine serumand 10 μg/mL gentamicin. The Dunning MAT LyLu rat tumor line is anandrogen-insensitive prostate tumor model that metastasizes to lymphnode and lungs in Copenhagen rats. MAT LyLu cells were maintained in thecomplete medium with added 250 nM dexamethasone.

Tumor Generation and Treatment: Dunning MAT LyLu cells were harvestedfrom culture flasks, and a 0.5 mL suspension containing from 2×10⁵ to2×10⁶ cells/mL was inoculated into the dorsal flank of adult Copenhagenrats (Harlan Sprague Dawley, Indianapolis, Ind.). The rats weighed250-300 g, and were 90-100 days old. Tumors were detected by palpatingthe skin around the site of injection starting 3 days following implantof the tumor cells (Lokeshwar et al. 1995a).

Drug Treatment In Vivo: Doxycycline and CMT-3 were dissolved in a 2%aqueous solution of a soluble form of carboxymethyl cellulose (SigmaCat. No. C-5678), and a fresh solution was made up daily. Rats weregavaged daily with 1 mL of the drug solution (concentrations specifiedbelow), or the vehicle (2% carboxymethyl cellulose). Tumor growth wasrecorded three times a week, and rats were weighed weekly. The effect ofvarious treatments on tumor growth was monitored over time usingcalipers, and the volume approximated to an ellipsoid (i.e.,volume=length×width×height×0.5236) (Lokeshwar et al. 1993b). Tumorgrowth rate was determined by regression analysis of tumor volume versustime, for each tumor-bearing rat. Mean tumor growth rate (time to reacha fixed volume) for each treatment group was then used to evaluate thestatistical significance of treatment efficacy using the INSTATstatistical program (Ravitz Software, San Diego, Calif.). Rats wereeuthanized once the tumor volume reached ≧10 cm³. At that time animalswere necropsied, tumors and lungs were removed and fixed in Bouin'sfixative. Macroscopic tumor foci on the lungs were counted under adissecting microscope.

EXAMPLE 2

Effect of CMTs on Prostate Cancer Cell Proliferation In Vitro

To determine the cytotoxicity of CMTs on prostate tumor cell lines,TSU-PR1 cells (and cells of other tumor cell lines) were exposed tovarious CMTs or to doxycycline for 24 hr or 48 hr in a complete medium.Cell viability (percent of live cells) was estimated by counting thecells following trypan blue staining. Cellular viability was alsoestimated by the tetrazolium dye reduction assay (MTT assay) (Lokeshwaret al. 1995b). Due to the aggressive proliferative capacity of cancercells, it is assumed that viable cells are actively proliferating.Therefore, the measurement of viability was used as an estimate ofproliferation. Results are expressed as Mean±SEM from three separateexperiments.

It was found that doxycycline and several CMTs reduced cellularviability and, hence, inhibited cell proliferation, in vitro. Inhibitionof cell proliferation was proportional to the concentration of the drugsand duration of exposure, but varied considerably from compound tocompound. In particular, CMT-2 and CMT-3 were significantly morecytotoxic than doxycycline. For the two human prostate cancer cell linesDU-145 and TSU-PR1, the 50% inhibition dose (IC₅₀) for various CMTsranged from 2.7 μg/mL (CMT-3, 48 hr exposure) to 120 μg/mL (CMT-6). TheIC₅₀ for CMT-2 was 5.7 μg/mL. CMT-5 was not inhibitory. Representativeresults are illustrated for the cell lines LNCaP, TSU PR1 and MAT LyLuin which a panel of CMTs was tested along with doxycycline (FIGS.2A-2C); and for the cell lines DU-145, PC-3, BPH-1 and FHS733 (a normalhuman fibroblast cell line) against which (FIGS. 3A-3D).

EXAMPLE 3A

Cytotoxic Effect of CMTs on MAT LyLu Cells In Vitro

Similar results were obtained when doxycycline and several CMTs (CMT-2,CMT-3, and CMT-6) were tested on the Dunning MAT LyLu cells in vitro.The Dunning MAT LyLu cells were exposed to the drugs for 24 hr or 48 hrbefore estimating the cellular viability. Cell viability was estimatedby trypan blue staining following exposure to the drugs. The results ofthese studies are summarized in FIG. 4A (24 hours) and FIG. 4B (48 hr).Data are presented as Mean±SEM from three separate experiments. CMT-3and CMT-2 were the most effective inhibitors of cell proliferation inthis assay.

EXAMPLE 3B

Effect of CMT-3 on Prostate Cancer Cell Proliferation In Vitro

Based on the data described above, it appeared that CMT-3 was the mostpotent (most cytotoxic) of all of the tested CMTs in vitro. To confirmthis observation, we compared the cytotoxicities of CMT-3 anddoxycycline for a panel of common human prostate cancer cell lines.Cytotoxicity was measured using the MTT assay (see above) and a cellularthymidine incorporation assay as described by Lokeshwar et al. (1995a).The 50% growth inhibition (GI₅₀) values obtained using both assays wereidentical. As shown in the data summarized in Table 3, below, CMT-3 wastwo- to eight-fold more cytotoxic than doxycycline.

TABLE 3 Cytotoxicity of CMT-3 and Doxycycline Against Prostate CancerCell Lines Cancer Cell Line Cytotoxicity GI₅₀ (μM) (Number of ReplicateExperiments) Doxycycline CMT-3 ALVA 101 (4) 36.6 ± 2.42 6.82 ± 0.75BPH-1 (3) 21.3 ± 5.4  10.53 ± 3.7  DU 145 (8) 43.6 ± 9.36 5.06 ± 1.18LNCaP(5) 13.86 ± 2.97  5.0 ± 2.1 MAT LyLu (7) 20.0 ± 6.4  5.2 ± 1.9 PC-3(5) 36.4 ± 2.5  10.56 ± 2.1  TSU PR-1 (5) 41.0 ± 11   14.74 ± 2.64 

Growth inhibition was calculated from linear regression of thedose-response curves generated for each experiment using log(dose) vs.cell proliferation (% of control). Correlation coefficient (r) wasalways ≧0.95 (negative). Results are presented as Mean±SEM of at leastthree GI₅₀ values calculated from each experiment.

EXAMPLE 4

Effect of CMTs on Invasive/Metastatic Potential of Prostate Cancer Cells

Treating prostate cancer cells with certain CMTs significantly inhibitedthe cells' ability to invade an artificial construct of tumor basementmembrane (Lokeshwar et al. 1996). In this method, 4×10⁵ tumor cells wereplated on the top chamber of the Boyden chemotaxis assay chambers(Costar Transwell plates). The bottom side of the chamber was a 12μm-pore polycarbonate filter layered with 0.5 mm thick layer ofMatrigel. The bottom well contained a chemoattractant, a serum-freeculture-conditioned medium from FHS 733 cells, a line of human fetallung fibroblasts (ATCC No. HTB-157). Cancer cells (4×10⁵) were plated inthe top wells of the plates. Doxycycline or CMTs were diluted in aserum-free medium to 5 μg/mL, and were added to both top and bottomchambers. The control wells contained only 0.1% dimethyl sulfoxide(DMSO, a diluent). After 48 hr incubation, MTT was added to both the topand bottom wells (0.5 mg/mL), and incubation was continued for 4 hr.Wells were then emptied, and the cells from the undersides of the filterwere pooled with those in the bottom wells with a filter tip. Thereduced MTT (formazan) from top and bottom wells was solubilized withDMSO overnight, and absorbance (O.D.) at 515 nm was measured. The ratioof the O.D. from the bottom wells to that of the total (i.e., O.D. oftop plus bottom wells) was taken as the invasive potential. Thisprocedure was consistently superior to that used in earlier reports(Albini et al. 1987), where cells on the bottom sides of the filterswere counted from several randomly chosen optical fields. The MTT assayprocedure also normalizes the coincident inhibition due to the cytotoxiceffects of the agents under investigation. Results presented are fromthree independent experiments.

The effect of CMT-3 and other tetracycline compounds on the invasivepotential of TSU-PR1 and the Dunning MAT LyLu cells was evaluated usingthe Matrigel assay. As shown in FIG. 5A, the ability of these compoundsto inhibit invasive activity varied significantly. CMT-3 was the mostpotent, and CMT-7 the least potent, inhibitor of the invasive/metastaticpotential of TSU-PR1 cells. The CMTs were surprisingly effective incomparison to a common tetracycline, doxycycline, which caused only amodest (8±1.8%) inhibition of Matrigel invasion of TSU-PR1 cells. Inparticular, the 50% inhibitory dose (IC₅₀) calculated for various CMTsvaried from 1.7±0.31 μg/mL for CMT-3 to >100 μg/mL for CMT-7.Doxycycline was not significantly inhibitory in TSU PR1 cells,(IC₅₀=27±4.3 μg/mL). The IC₅₀ values for CMT-3 and doxycycline withrespect to three other invasive human prostate cancer cell lines (DU145, PC-3, and ALVA 101) were in the same concentration ranges as forTSU PR1 (data not shown).

In the Dunning cells, both CMT-2 and CMT-3 equally inhibited Matrigelinvasion. See FIG. 5B. Moreover, doxycycline also significantlyinhibited (68±4.2%) the invasive/metastatic potential of the Dunning MATLyLu cells over a 48 hr period. Continuous presence of the drugs wasneeded to achieve significant inhibition of invasive potential.Forty-eight hour pretreatment with CMT-3, followed by deletion of thedrugs in the invasion chamber, had only a moderate effect on theinvasive potential.

The 48 hr invasion assay had the following invasion indices in thecontrol wells (0.1% DMSO): 22±8.3 for TSU-PR1 cells and 17±4.2% for MATLyLu cells.

EXAMPLE 5

Effect of CMT-3 on MAT LyLu Tumor Growth and Lung Metastasis

CMT-3 and doxycycline were tested for antitumor activity in vivo. Inthis series of experiments, daily gavage of drugs was started on thesame day on which tumor cells were implanted in the test animals. Tumorgrowth was initiated by sub cutis injection of 1×10⁶ tumor cells.

Tumors were palpable (≧0.1 cm³) in more than 50% of injected animals byday 6, and in 100% of the animals by day 12. Tumors rapidly increased involume, reaching >10 cm³ by 15 days post-implant. Tumor growth rate, asdetermined by the time to reach a volume of 3 cm³, did not varysignificantly between rats treated with doxycycline or CMT-3 atconcentrations of 20 mg/kg or 40 mg/kg, and rats given the vehicle alone(2% solution of carboxymethyl cellulose). Regression analysis of tumorvolumes showed no significant difference in the primary tumor growthbetween the control group and the doxycycline- and the CMT-3-treatedgroups. Specifically, the time period from injection of cells to agrowth of 3 cm³ tumor was 13.57±2.12 days in the control group and14.0±1.9 days in the CMT-3-treated group. All of the tumors, from thecontrol group. as well from the drug-treated group developed highlynecrotic centers as the tumors grew to 10 cm³ or larger.

Metastatic tumor foci (MTF) were visible in lungs fixed in Bouin'sfixative. Most of the MTF were less than 1 mm in diameter in all thetreatment groups. FIG. 6 shows the number of metastatic foci in thelungs (Mean±SEM). As illustrated in FIG. 6, the control group showed59.5±13.9 MTF/rat (Mean±SD) and only 39.7±17.2 or 43.6±18.8 MTF/rat inlow dose (20 mg/kg) and high dose (40 mg/kg) doxycycline-treated group,respectively. The high-dose CMT-3 group (10 mg/rat, i.e., 40 mg/kg) hadthe most significant reduction in MTF, 28.9±15.4 MTF/rat, a 51%reduction in MTF relative to the control group (p<0.01, Tukey KramerMultiple comparison test). Histological examination of the lung sectionsdid not reveal any apparent differences in tumor foci among varioustreatment groups.

EXAMPLE 6

Effect of Pretreatment on Tumor Growth and Metastasis

In another series of experiments, we examined whether predosing hostanimals with the drugs would affect tumor growth and metastasis. Dailygavage of control or drugs (doxycycline or CMT-3; 40 mg/kg) was begun 7days prior to the injections of the MAT LyLu tumor cells (2×10⁵cells/animal) and continued for a total of 21 days.

Using this schedule, we found a decrease in tumor incidence and tumorgrowth rate. Tumor growth rates were calculated from thrice-weeklymeasurement of tumor volume with linear regression analysis oflog-transformed volume measurements (Dudak et al. 1996). Tumor growthrates determined by this method were then tested by Tukey-Kramermultiple comparison test and were found to be significantly differentfrom the control group.

As shown in FIG. 7A, tumor incidence was >90% in the control anddoxycycline-treated groups in three independent experiments.Surprisingly, however, tumor incidence in CMT-3-treated rats varied from28% (2/7) to 85% (6/7) in four separate experiments. Thus, tumorincidence was significantly lower (by 55%±18) than for the control ordoxycycline-treated groups. The rats with no primary tumor incidenceremained tumor-free for up to six months, at which time they wereeuthanized. No histologically identifiable tumor foci were observed,whether at the site of injection or in the lungs. Furthermore, apreparation of CMT-3 of greater purity reduced the tumor incidence to43% (data not shown). Thus, only CMT-3, and, not the commerciallyavailable tetracycline, doxycycline, produced significant inhibition ofprimary tumor incidence.

In addition, among the rats in the CMT-3-treated group that developedmeasurable tumors (≦50%), the tumor growth rate (time to reach tumorvolume of 3 cm³) was significantly slower (20.2±3.5 days) than in eitherthe control group (15.9±2.0) or the doxycycline-treated group (16.7±1.9days). It should be emphasized that the tumor growth rate was calculatedfrom the subset of animals in which the tumors were measurable. Thus,the overall effect of CMT-3 on tumor growth was greater than thatsuggested by these data.

Applicants were especially surprised by the apparent remission ofpalpable tumors (i.e., tumor resorption; reduction of tumor size) inrats treated with CMT-3 (30%) or doxycycline (20%) in two separateexperiments. In those animals, tumors were palpable by 8-10 days afterinjection at the primary site, but the tumors did not increase involume, and disappeared 4-7 days later. These rats with no primary tumorincidence remained tumor-free and healthy up to eight months, at whichtime they were euthanized.

CMT-3 also inhibited tumor metastasis by predosing the animals withCMT-3. See FIG. 7B. This effect was comparable to that observed withoutpredosing. The MTF in treated animals was 46.3±6.7 in the CMT-3-treatedgroup versus 74.2±6.4 in controls, a 37.5% decrease. Data presented areexpressed as Mean±SD from all the animals in which tumors grew to a sizeof ≧10 cm³. The lungs removed from tumor-free animals were free ofhistologically recognizable metastasis.

None of the typical adverse effects of conventional chemotherapeuticdrug treatments, such as irritability, hypersensitivity to light, hairloss, or diarrhea were observed in association with the tetracyclinetreatments described hereinabove. As a gross measure of injury to normaltissue plausibly caused by doxycycline or CMT-3, animals were weighedbefore, during, and after treatment, and all changes were examined. Inall experiments, none of the animals showed a significant weight loss;instead, there was a 3% (control group) to 12% (CMT-3 treated group)gain in body weights as of the time the experiment was terminated(6-month post-treatment observation period). Thus, the method of theinvention has a substantial advantage over such conventional anti-cancertherapies inasmuch as less, and preferably virtually no, toxicity tonormal tissue is seen at cancer-inhibitory tetracycline dosages.

EXAMPLES 7-10

In Examples 7-10 below, the following materials and methods wereemployed:

Cell Culture and Maintenance: C8161 cells, a human melanoma cell line,were maintained in Dulbecco's modified Eagle's medium (DMEM; GIBCO,Grand Island, N.Y.), supplemented with 10% heat-inactivated fetal bovineserum (FBS; GIBCO) and 0.1% gentamicin sulfate. These cells wereroutinely screened for Mycoplasma contamination using the GenProbe RapidDetection System (Fisher Scientific; Chicago, Ill.).

Chemically Modified Tetracyclines: Fresh stock solutions of thechemically modified tetracycline compounds (2 mg/mL) were prepared foreach experiment by hydrating in 2% dimethyl sulfoxide (DMSO)/water pH10, then adjusting to pH 7.4 using 1.0 M HCl.

EXAMPLE 7

Effect of Chemically Modified Tetracyclines on Cell Proliferation

One hundred thousand (1×10⁵) C8161 cells were seeded per well in each ofthree 24-well culture dishes in the presence of either DMSO (0.05%;Sigma Chem. Co.; control) or 50 μg/mL of doxycycline or the other CMTs(three wells on each plate per compound). Cells were harvested from thefirst plate after 24 hr with 2 mM EDTA in phosphate buffered saline(PBS, minus divalent cations), from the second plate after 48 hr andfrom the third plate after 72 hr. The doubling time for C8161 cells inthe presence of each compound was then determined and compared to theDMSO treated cells (control).

The effect of CMTs on the proliferation of C8161 cells on plastic isreported in Table 4, below:

TABLE 4 Effect of Tetracycline Compounds on C8161 Cell ProliferationCompound Doubling Time DMSO 22 hr CMT-1 24 hr CMT-3 28 hr CMT-4 22 hrCMT-6 24 hr CMT-7 24 hr CMT-8 26 hr Doxycycline 22 hr

As shown, CMT-4 and doxycycline did not affect the doubling time ofC8161 cells on plastic; CMT-1, CMT-6 and CMT-7 slightly increased thedoubling time of the cells (approximately 9%), and CMT-8 and CMT-3increased the doubling time by approximately 18% and 27%, respectively.

EXAMPLE 8

Gelatin-Incorporated SDS-Polyacrylamide Gel Electrophoresis (Zymography)

Six hundred thousand (6×10⁵) C8161 cells were seeded per well in a12-well dish coated with a laminin/collagen IV/gelatin matrix in DMEMplus Mito+ and 0.1% gentamicin sulfate. After approximately 1 hrincubation to allow the cells to attach, 50 μg/mL of either doxycyclineor one of the other CMTs was added per well and the dish then placed ina 37° C. humidified 5% CO₂ incubator. After 24 hr, the supernatants wereremoved and centrifuged to remove any cells or debris. One volume ofLaemmli sample buffer minus reductant was added to two volumes of themedium, and this sample electrophoresed without prior heating or boilingon a 10% SDS-PAGE containing 0.1% gelatin (samples normalized based onthe same number of cells per volume of medium per time of incubation).After electrophoresis, the gels were washed with gentle shaking at roomtemperature for 30 min in 50 mM Tris-HCl (pH 7.5) plus 2.5% TRITON® X100plus 50 μg/mL of the corresponding CMT used in the original treatment.The gel was then placed in incubation buffer (50 mM Tris-Cl/10 mMCaCl₂/150 mM NaCl/0.05% NaN₃) also containing 50 μg/mL of thecorresponding CMT, and incubated at 37° C. for 20-24 hr. The gels werestained with Coomassie BBR-250, then destained with 10% methanol/10%acetic acid in water until the wash remained clear. A photographicnegative of the gel was digitized using a video camera and Snappy VideoSnapshot system (Play Inc., Rancho Cordova, Calif.). An integrateddensity was determined for each of the cleared zones of proteolysisusing the software ImagePC8α (freely available from the NationalInstitutes of Health), and the corresponding changes in gelatinolyticactivities reported compared to the control samples normalized to avalue of 1.0.

The relative amount of gelatinolytic enzyme activity in the conditionedmedium from C8161 cells treated with these compounds was measured byzymography (data not shown), and quantified against the amount ofactivity in the control samples by densitometric analysis, as shown inTable 5, below:

TABLE 5 Densitometric Analysis of Zymograms Compound Gelatinase AGelatinase B Control 1.00* 1.00* Doxycycline 0.26 0.40 CMT-1 0.24 0.22CMT-2 0.89 1.51 CMT-3 0.22 0.03 CMT-4 0.75 0.12 CMT-6 0.07 0.51 CMT-70.17 1.22 CMT-8 0.70 1.05 *Integrated density of digitized photographicnegative normalized to a value of 1.00 for the control samples using theimage analysis software ImagePCα (NIH).

Treatment of C8161 cells with each of these compounds resulted in adecrease in extracellular levels of gelatinase A activity (Table 5),ranging from 11% to 93%(CMT-2<CMT-4<CMT-8<doxycycline<CMT-1<CMT-3<CMT-7<CMT-6). Cells treatedwith five of the compounds also resulted in a decrease in extracellularlevels of gelatinase B, from 49% to 97%(CMT-6<doxycycline<CMT-1<CMT-4<CMT-3). Treatment with three of the CMTsresulted in an increase in extracellular levels of gelatinase B (5 to51%; CMT-8<CMT-7<CMT-2).

EXAMPLE 9

In Vitro Invasion Assay

The in vitro invasive potentials of the control (DMSO only) andchemically-modified tetracycline treated cells were measured using amodified Boyden chamber as previously described (Membrane InvasionCulture System, MICS). An intervening barrier consisting of apolycarbonate filter containing 10 μm pores (Osmonics, Livermore,Calif.) coated with a defined matrix of human laminin/collagenIV/gelatin (Sigma, St. Louis, Mo.) was used for these studies. C8161human melanoma cells were seeded into the upper wells of the chamber inDMEM containing Mito+ (Collaborative Biomedical, Bedford, Mass.; i.e.,serum-free medium) and allowed to attach at 37° C. in a humidified 5%CO₂ incubator. Doxycycline or individual CMTs were then added todifferent wells of the chamber at 3, 20 or 50 μg/mL 2 hr post-seedingand daily during the assay. After 48 hr, the cells were removed from thelower wells and the number of invasive cells was determined and comparedto the original number of cells seeded into the upper wells. Whereappropriate, data were corrected for proliferation during the period ofthe in vitro assay.

The effects of doxycycline and the CMTs at three differentconcentrations (range: 3 μg/mL; 20 μg/mL; 50 μg/mL) on the in vitroinvasive potential of C8161 cells are shown in FIG. 8. These data aresummarized in Table 6, below.

TABLE 6 Inhibition of Invasive Potential of C8161 Cells Through aLaminin/Collagen IV/Gelatin Matrix-Coated Filter Compound PercentInhibition Type of Inhibition Doxycycline 12-79% dose dependent CMT-126-58% dose-dependent CMT-2 ˜10% non-dose dependent CMT-3 35-74% dosedependent CMT-4 ˜20% non-dose dependent CMT-6  8-50% dose dependentCMT-7 15-55% non dose-dependent CMT-8 ˜12% non-dose dependent

EXAMPLE 10

In Vivo Metastasis Assay

Immunosuppressed mice (athymic nude/nude SCID females from HarlanSprague Dawley) were housed in autoclaved cages with microisolator tops,and all manipulations of the animals were done in a laminar flow hoodafter wiping down both the hood, gloves and cages with ABQ sterilant.The mice were fed sterile Pico Lab Chow (Purina) and autoclaved St.Louis tap water. Doxycycline or the CMTs were administeredintrα-gastrically daily to the mice in sterile water containing 2%carboxymethyl cellulose via sterile, disposable animal feeding needles(Poper & Sons Cat #9921; 20 g×1.5″), seven days a week between 7:00 and8:00 am. The compounds and control (sterile water plus 2% carboxymethylcellulose) were kept stored at −80° C. wrapped in aluminum foil toprevent any light induced changes, and each day's supply was thawed justprior to use.

Four compounds were tested for their effects on the metastatic potentialof C8161 cells injected intravenously via the tail vein: CMT-1, CMT-3,and CMT-7 at 40 and 100 mg/kg, CMT-8 at 40 mg/kg, compared to thecontrol. The concentration of the compounds in the vials used to givethe 100 mg/kg doses were 2.5 times that in the 40 mg/kg dose so thatapproximately the same volume was used in both cases, approximately 0.5mL/animal. The experiments started with nine animals per group at day−4. On day zero, 2×10⁵ C8161 cells in cold Hank's Balanced Salt Solution(HBSS) were injected intravenously via tail vein inoculation. Theexperiment was continued for an additional 24 days, at which time theanimals were sacrificed and their lungs removed and fixed in a solutionof Bouins/formaldehyde (5 parts:1 part). Tumors were quantitated on theentire surface of the lungs by rotating the lungs and counting thetumors on each lobe using a 6× magnifying glass. Statistical analysiswas performed using the statistical package of Microsoft's Excelspreadsheet software.

The effects of CMT-1, CMT-3, CMT-7 and CMT-8, at two differentconcentrations, on the metastatic potential of C8161 cells in SCID miceare presented in Table 7, below:

TABLE 7 Effect of CMTs on the Metastatic Potential of C8161 Cells inSCID Mice Number of Lung Number of Compound Dosage Metastases/Mouse*Range Mice (n) p value^(†) Control  0.5 mL vehicle^(‡) 231 ± 49  120to >250 7 CMT-1  40 mg/kg 129 ± 102  3 to >250 9 0.023 100 mg/kg 240 ±28  165 to >250 5 0.647 CMT-3  40 mg/kg 111 ± 105  4 to >250 9 0.033 100mg/kg 176 ± 82   9 to >250 5 0.141 CMT-7  40 mg/kg 136 ± 107  63 to >2506 0.169 100 mg/kg 166 ± 101  11 to >250 5 0.157 CMT-8  40 mg/kg 186 ±60  124 to >250 7 0.184 100 mg/kg (see note below) *Average ± SEM. ^(†)p< 0.05 compared to control is considered a significant difference.^(‡)Sterile water containing 2% carboxymethyl cellulose. Note: Thisgroup of mice was the first to be treated by gavage, and did not survivedue to technical difficulties.

Oral gavaging of the animals with CMT-1 or CMT-3 significantly reducedthe number of lung metastases in the SCID mouse population whenadministered daily at 40 mg/kg (p<0.05), but did not significantlyreduce the number of lung metastases at the 100 mg/kg dosage. Thecompounds CMT-7 and CMT-8 did not significantly reduce the number oflung metastases at either dosage (p>0.05).

EXAMPLE 11

Differential Cytotoxicity of Tetracycline Compounds Against Cancer Cells

Several cancer cell lines and normal cells were examined in vitro todetermine whether tetracycline compounds produce cytotoxic (cellkilling) effects. A conventional assay for viability of cultured cellsbased on redox activity in the cells was employed (Pagé et al. 1993).The assay uses a redox dye Alamar Blue, which is a fluorogenic indicatordye that is converted to a fluorescent red product only when cells arecarrying out electron transport activity, a widely accepted criterion ofviability.

Cultured cells from three prostate tumor cell lines, LNCAP, DU-145, andPC-3, and normal prostate stromal cells obtained by biopsy of a normal37-year old man, were allowed to grow to confluence in multiwellmicroplates to minimize differences in proliferative activity among thecell types at the time of exposure to the test compound. CMT-3 was addedto the wells at concentrations of 10 μM and 20 μM (50 μM also in thecase of DU-145). At the start of the experiment, and again at 1, 2, and3 days, selected wells were incubated with the indicator dye for 3 hr,and the fluorescence was measured on a Cytofluor 2300 fluorescentmicroplate reader. The results are summarized in FIGS. 9A-9D.

Normal prostate stromal cells showed virtually no change in fluorescenceover the three-day period, regardless of CMT-3 dose (FIG. 9A). A slightincrease on day 1 in the stromal cells not exposed to CMT-3 supportedour intention to avoid introducing the complication of markeddifferences in proliferative activity among the different cell beingstudied. LNCAP cells proved to be exquisitely sensitive to CMT-3 evenunder these conditions of limited proliferative activity: cytotoxicityis complete within 24 hr at 20 μM, and nearly complete at that time in10 μM CMT-3 (FIG. 9B). PC-3 tumor cells are somewhat less susceptible toCMT-3, which induces about 25% cytotoxicity at 24 hr, 50% at 48 hr and75% after 72 hr (FIG. 9C). DU-145 tumor cells are less sensitive, withsome cytotoxicity appearing after 72 hr at 20 μM (FIG. 9D). DU-145 cellsshow significant cytoxicity at 50 μM, which gradually develops over thecourse of the 72 hr test period. Normal cells exposed to 50 μM CMT-3show no comparable cytotoxicity (data not shown).

It is to be noted that marked differences in cellular morphology wereobserved between the normal stromal cells and the tumor cells afterexposure to CMT-3: no significant changes could be seen in the normalstromal cells, whereas all of the tumor cell types became progressivelyvacuolated.

EXAMPLE 12

Cytotoxicity of Tetracycline Compounds Against Cancer Cells

A panel of chemically modified tetracycline compounds and doxycyclinewere tested for cytotoxicity against two carcinomas: COLO 205 (a humancolon carcinoma-derived cell line) and the E-10 clone of MDA-MB-231 (ahuman breast carcinoma-derived cell line). Cytoxicity was measured usinga conventional assay for viability with a tetrazolium salt (MTS). Cellswere grown in media containing the CMTs at concentrations ranging from 5μM to 100 μM for two days. The cells were then incubated with the MTSfor several hours before removal of the supernatant medium fordetermination of formazan, which is generated only by the viable cells.The results are presented in Tables 8A and 8B below. Results areexpressed as percent cytotoxicity, which is simply the percent declinein measured formazan concentration in the sampled medium. Two differentlots of CMT-3 were tested, designated “A” and “B”.

TABLE 8A Cytotoxic Effect of CMTs on COLO 205 Cells In Vitro Compound 5μM 10 μM 20 μM 50 μM 100 μM CMT-1 0 4 32.5 63.8 82.8 CMT-2 2.7 4.5 0 716.3 CMT-3 17 28.8 52 79 85.6 CMT-3 25.9 38.9 37 86.7 86.7 CMT-4 1.8 6.825.3 73.3 81.9 CMT-5 0 0 0 0 0 CMT-6 1.7 0 0 0 22.2 CMT-7 0 0 0 0 29.4CMT-8 0 20.5 35.5 44.4 50 Doxycycline 0 0 45.7 58.8 66.5

TABLE 8B Cytotoxic Effect of CMTs on E-10 Cells In Vitro Compound 5 μM10 μM 20 μM 50 μM 100 μM CMT-1 7.6 0 0 2.2 7.6 CMT-2 1.5 3.8 0.8 0 2.3CMT-3 0 0 4.15 3 13.9 CMT-3 0 1.8 8.5 17.7 47 CMT-4 9 16.7 0.8 0 0 CMT-50 0 0 0 0 CMT-6 0 6 3 7.6 16.7 CMT-7 0 0 0 0 8.3 CMT-8 0 0 0 0 0Doxycycline 0 0 4.5 24.2 29.5

The above data show that CMT-3 is the most potent of the testedcompounds, with CMT-1, CMT-4, and CMT-8 also producing significantcytotoxicity. Doxycycline also produced some cytotoxicity.

EXAMPLE 13

Cytotoxicity of Tetracycline Compounds Against Cancer Cells

An investigation of the cytotoxicity of chemically modified tetracyclinecompounds for cancer cells was undertaken to determine involvement ofprogrammed cell death (apoptosis) (see Duke et al. 1986). MAT LyLu cellswere incubated with various concentration of CMT-3 and doxycycline.Conditioned media were assayed for soluble nucleosomes, resulting frominternucleosomal DNA strand breaks, using the Cell Death Detection ELISAPlus kit from Boehringer Mannheim GmbH (Catalog No. 1774425) accordingto the manufacturer's instructions. In the dosage-dependence trials,cells were incubated with the tetracycline compounds for 48 hr, and thenmedia were collected. In the time trials, the cells were exposed to thetetracycline compounds for specific periods of time from 0 to 48 hr, andif appropriate were maintained in fresh media until 48 hr from the startof the experiment at which time media were collected. The experimentswere performed three times, and representative data are shown in FIGS.10A and 10B.

As shown in FIG. 10A, a dose-dependent increase in soluble nucleosomeswas observed in the MAT LyLu cells treated with CMT-3, but not in thecells treated with doxycycline. In fact, programmed cell death inductionwas insignificant even at a doxycycline concentration exceeding the 50%growth inhibiting dose (GI₅₀) as determined by proliferation assays asdescribed above. The amount of soluble nucleosomes released into themedia was directly proportional to the fractions of apoptotic cells, asdetermined microscopically in select cases. Furthermore, as shown inFIG. 10B, a brief period of exposure to CMT-3 (4 hr at 5 μg/mL or 10μg/mL) was sufficient to elicit a putative programmed cell deathresponse in the MAT LyLu cells, but no such response was observed forthe doxycycline-treated cells (data not shown).

Thus, while there have been described what are presently believed to bethe preferred embodiments of the present invention, those skilled in theart will realize that other and further embodiments can be made withoutdeparting from the spirit of the invention, and it is intended toinclude all such further modifications and changes as come within thetrue scope of the claims set forth herein.

EXAMPLE 14

Effect of CMT-3 on Kaposi's Sarcoma

CMT-3 was administered orally, once daily, in a dose escalating fashionto 3-6 subjects per dose cohort. Patients with symptomatic visceralKaposi's Sarcoma or severe tumor-associated edema were excluded.Antiretroviral therapy was permitted but not required. Study endpointswere grade 3 or grade 4 toxicity or progression of disease. Preliminaryanalysis was performed.

Eighteen patients received CMT-3 in dosing cohorts of 25, 50 and 70mg/m²/day. The average age was 39 years (range, 25-57). Median CD4 countwas 352 cells/mm³ (range, 6-694). Prior therapy for Kaposi's Sarcoma wasreported by 16 (89%) patients; prior antiretroviral therapy by 16 (89%)patients. Half of the patients had ≧50 lesions at baseline. Grade 3 orhigher events, which were possibly/definitely related to study drug,were reported by 6 patients and consisted of photosensitivity, increasedpain, headache, rash and pruritis. Photosensitivity was dose-related; nophotosensitivity was observed in the 25 mg/m²/day cohort. Partialresponses were observed in 7 patients: 2 in the 25 mg/m²/day cohort, 2in the 50 mg/m²/day cohort and 3 in the 70 mg/m²/day cohort.

CMT-3, when administered orally once daily in the treatment ofHIV-related Kaposi's Sarcoma, is well tolerated, particularly the 25mg/m²/day dose.

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What is claimed is:
 1. A method of inhibiting a sarcoma in a mammal,comprising administering to the mammal a sarcoma-inhibitory amount of6-demethyl-6-deoxy-4-de(dimethylamino)tetracycline (CMT-3).